Selective CD28 Blockade Results in Superior Inhibition of Donor-Specific T Follicular Helper Cell and Antibody Responses Relative to CTLA4-Ig.

Donor-specific antibodies (DSAs) are a barrier to improved long-term outcomes after kidney transplantation. Costimulation blockade with CTLA4-Ig has shown promise as a potential therapeutic strategy to control DSAs. T follicular helper (Tfh) cells, a subset of CD4+ T cells required for optimal antibody production, are reliant on the CD28 costimulatory pathway. We have previously shown that selective CD28 blockade leads to superior allograft survival through improved control of CD8+ T cells relative to CTLA4-Ig, but the impact of CD28-specific blockade on CD4+ Tfh cells is unknown. Thus, we identified and characterized donor-reactive Tfh cells in a murine skin transplant model and then used this model to evaluate the impact of selective CD28 blockade with an anti-CD28 domain antibody (dAb) on the donor-specific Tfh cell-mediated immune response. We observed that the anti-CD28 dAb led to superior inhibition of donor-reactive CXCR5+ PD-1high Tfh cells, CD95+ GL7+ germinal center B cells and DSA formation compared with CTLA4-Ig. Interestingly, donor-reactive Tfh cells differentially upregulated CTLA4 expression, suggesting an important role for CTLA4 in mediating the superior inhibition observed with the anti-CD28 dAb. Therefore, selective CD28 blockade as a novel approach to control Tfh cell responses and prevent DSA after kidney transplantation warrants further study.

Effects of Lung Cotransplantation on Cardiac Allograft Tolerance Across a Full Major Histocompatibility Complex Barrier in Miniature Swine.

A 12-day course of high-dose tacrolimus induces tolerance of major histocompatibility complex-mismatched lung allografts in miniature swine but does not induce tolerance of heart allografts unless a kidney is cotransplanted. To determine whether lungs share with kidneys the ability to induce cardiac allograft tolerance, we investigated heart-lung cotransplantation using the same induction protocol. Hearts (n = 3), heart-kidneys (n = 3), lungs (n = 6), and hearts-lungs (n = 3) were transplanted into fully major histocompatibility complex-mismatched recipients treated with high-dose tacrolimus for 12 days. Serial biopsy samples were used to evaluate rejection, and in vitro assays were used to detect donor responsiveness. All heart-kidney recipients and five of six lung recipients demonstrated long-term graft survival for longer than 272 days, while all heart recipients rejected their allografts within 35 days. Tolerant recipients remained free of alloantibody and showed persistent donor-specific unresponsiveness by cell-mediated lympholysis/mixed-lymphocyte reaction. In contrast, heart-lung recipients demonstrated rejection of both allografts (days 47, 55, and 202) and antidonor responsiveness in vitro. In contrast to kidneys, lung cotransplantation leads to rejection of both heart and lung allografts, indicating that lungs do not have the same tolerogenic capacity as kidneys. We conclude that cells or cell products present in kidney, but not heart or lung allografts, have a unique capacity to confer unresponsiveness on cotransplanted organs, most likely by amplifying host regulatory mechanisms.

A novel approach to measuring cell-mediated lympholysis using quantitative flow and imaging cytometry.

In this study, we established a novel isotope-free approach for the detection of cell-mediated lympholysis (CML) in MHC defined peripheral blood mononuclear cells (PBMCs) using multiparameter flow and imaging cytometry. CML is an established in vitro assay to detect the presence of cytotoxic effector T-lymphocytes precursors (CTLp). Current methods employed in the identification of CTLp in the context of transplantation are based upon the quantification of chromium ((51)Cr) released from target cells. In order to adapt the assay to flow cytometry, primary porcine PBMC targets were labeled with eFluor670 and incubated with major histocompatibility complex (MHC) mismatched effector cytotoxic lymphocytes (CTLs). With this method, we were able to detect target-specific lysis that was comparable to that observed with the (51)Cr-based assay. In addition, the use of quantitative cell imaging demonstrates the presence of accessory cells involved in the cytotoxic pathway. This innovative technique improves upon the standard (51)Cr release assay by eliminating the need for radioisotopes and provides enhanced characterization of the interactions between effector and target cells. This technique has wide applicability to numerous experimental and clinical models involved with effector-cell interactions.

Kidney-induced cardiac allograft tolerance in miniature swine is dependent on MHC-matching of donor cardiac and renal parenchyma.

Kidney allografts possess the ability to enable a short course of immunosuppression to induce tolerance of themselves and of cardiac allografts across a full-MHC barrier in miniature swine. However, the renal element(s) responsible for kidney-induced cardiac allograft tolerance (KICAT) are unknown. Here we investigated whether MHC disparities between parenchyma versus hematopoietic-derived "passenger" cells of the heart and kidney allografts affected KICAT. Heart and kidney allografts were co-transplanted into MHC-mismatched recipients treated with high-dose tacrolimus for 12 days. Group 1 animals (n = 3) received kidney and heart allografts fully MHC-mismatched to each other and to the recipient. Group 2 animals (n = 3) received kidney and heart allografts MHC-matched to each other but MHC-mismatched to the recipient. Group 3 animals (n = 3) received chimeric kidney allografts whose parenchyma was MHC-mismatched to the donor heart. Group 4 animals (n = 3) received chimeric kidney allografts whose passenger leukocytes were MHC-mismatched to the donor heart. Five of six heart allografts in Groups 1 and 3 rejected <40 days. In contrast, heart allografts in Groups 2 and 4 survived >150 days without rejection (p < 0.05). These data demonstrate that KICAT requires MHC-matching between kidney allograft parenchyma and heart allografts, suggesting that cells intrinsic to the kidney enable cardiac allograft tolerance.

Induction of cardiac allograft tolerance across a full MHC barrier in miniature swine by donor kidney cotransplantation.

We have previously shown that tolerance of kidney allografts across a full major histocompatibility complex (MHC) barrier can be induced in miniature swine by a 12-day course of high-dose tacrolimus. However, that treatment did not prolong survival of heart allografts across the same barrier. We have now tested the effect of cotransplanting an allogeneic heart and kidney from the same MHC-mismatched donor using the same treatment regimen. Heart allografts (n = 3) or heart plus kidney allografts (n = 5) were transplanted into MHC-mismatched recipients treated with high-dose tacrolimus for 12 days. As expected, all isolated heart allografts rejected by postoperative day 40. In contrast, heart and kidney allografts survived for >200 days with no evidence of rejection on serial cardiac biopsies. Heart/kidney recipients lost donor-specific responsiveness in cell-mediated lympholysis and mixed-lymphocyte reaction assays, were free of alloantibody and exhibited prolonged survival of donor, but not third-party skin grafts. Late (>100 days) removal of the kidney allografts did not cause acute rejection of the heart allografts (n = 2) and did not abrogate donor-specific unresponsiveness in vitro. While kidney-induced cardiac allograft tolerance (KICAT) has previously been demonstrated across a Class I disparity, these data demonstrate that this phenomenon can also be observed across the more clinically relevant full MHC mismatch. Elucidating the renal element(s) responsible for KICAT could provide mechanistic information relevant to the induction of tolerance in recipients of isolated heart allografts as well as other tolerance-resistant organs.

Avoiding infrainguinal bypass wound complications in patients with chronic renal insufficiency: the role of the anatomic plane.

OBJECTIVE: To study the factors leading to wound problems in patients with chronic renal insufficiency (CRI) with emphasis on subcutaneous vs. deep placement of grafts. METHODS: The outcomes of patients undergoing an infrainguinal bypass with preoperative CRI (serum creatinine > or = 2.0 mg/dl) were reviewed. Surgical site infection (SSI) was classified as superficial or deep according to the Centres for Disease Control standards. RESULTS: Forty-two patients underwent a total of 47 infrainguinal bypasses for ischaemic rest pain or tissue loss. The graft location was partially or predominantly subcutaneous in 21 limbs (Group I) and 26 grafts were positioned in the anatomic or subfascial planes (Group II). In Group I, seven early (< 30 days postoperative), one intermediate (4-6 weeks postoperative), and one late (> 6 weeks postoperative) SSI's were found (9/21, 43%). In three of these patients the graft was exposed and two required removal. In contrast, only two early and one intermediate SSI's (3/26, 12%) were noted in Group II (p = 0.02). A logistic regression analysis, with twelve possible covariables wound healing, confirmed the subcutaneous location to be the only controllable factor significantly predicting SSI (relative risk = 11.6, p = 0.01). CONCLUSIONS: The infrainguinal bypass in patients with CRI is associated with a high incidence of wound complications. In our retrospective series, the presence of a vascular conduit in the subcutaneous plane was connected with a higher rate of SSI. Despite the growing trend toward the use of the in situ bypass, CRI may represent a circumstance where deeply placed grafts should be used preferentially.

The durability of different reconstructive techniques for atherosclerotic renal artery disease.

PURPOSE: Among various surgical techniques for renal artery reconstruction (RAR), anatomic aortorenal bypass has been the preferred standard. Yet concern regarding origin of the bypass from a diseased aorta and desire to avoid a major aortic operation in these patients who are often at poor risk has led to increasing use of extraanatomic bypass grafting, particularly hepatorenal and splenorenal bypass. This study was conducted to compare the safety and long-term performance of these different techniques of renal artery reconstruction. METHODS: We reviewed a 15-year (1976 to 1991) experience with 323 surgical RAR performed in 285 patients with atherosclerotic renovascular disease. Long-term patency and survival rates were analyzed by life-table methods. Variables potentially affecting early failure of the RAR and perioperative and late mortality rates were examined by Cox proportional hazards models. RESULTS: Diffuse atherosclerosis characterized the patients' clinical profile. Clinically evident coronary artery disease was present in 54% of patients, and some degree of renal insufficiency was present in 60%. Ninety-five percent of patients had hypertension with poor control of hypertension seen in 50%. Aortic disease necessitated combined aortic grafting and RAR in 43% of the study group. Various techniques of RAR were used as follows: endarterectomy or patch angioplasty, 8.5%; extraanatomic bypass grafting, 37% (hepatorenal, 62; splenorenal, 52; iliorenal, 7); and aortorenal bypass grafting, 54% (native aorta, 34; combined aortic graft and RAR, 140). Early failure of the RAR occurred in 5% of cases, and the operative mortality rate for the entire cohort was 5.6%. Median follow-up duration was 9.4 years. A comparison of early and late patency for the major types of RAR revealed equivalent (p = 0.44) performance of aortorenal and extraanatomic bypass grafting. Perioperative complications occurred more frequently (p < 0.02) in patients undergoing combined operations. The cumulative 5-year survival rate for all patients was 75%. CONCLUSIONS: Because extraanatomic bypass grafting can provide long-term results equivalent to aortorenal bypass grafting, the choice among techniques for RAR in patients with diffuse atherosclerosis should be based on both technical and operative safety considerations, rather than adherence to aortorenal bypass grafting as an inherently superior technique.